Method of dyeing fabric using microorganisms

ABSTRACT

This invention relates to a method for dyeing fabrics, yarns and fibers using microorganisms whereby the adsorption of dye-containing microorganisms onto textile fibers is improved using carbon sources above a threshold concentration. Dye molecules contained within the microorganism are released from the microorganism and fixed directly and locally to the textile fibers using a heat treatment step. Said heat treatment also deactivates the carrier microorganisms. Single or multiple microorganism species, and single or multiple dyes produced by said single or multiple microorganism species may create a variety of textile colors. Suitable synthetic dyes may also be added before, during or after microorganisms have produced dyes but before the dye-releasing heat treatment step.

This invention relates to a method for dyeing fabrics, yarns and fibersusing microorganisms whereby the adsorption of dye-containingmicroorganisms onto textile fibers is improved using carbon sourcesabove a threshold concentration. Dye molecules contained within themicroorganism are released from the microorganism and fixed directly andlocally to the textile fibers using a heat treatment step. Said heattreatment also deactivates the carrier microorganisms. Single ormultiple microorganism species, and single or multiple dyes produced bysaid single or multiple microorganism species may create a variety oftextile colors. Suitable synthetic dyes may also be added before, duringor after microorganisms have produced dyes but before the dye-releasingheat treatment step.

Dye Production

Most contemporary fabric dyes are synthesized chemically and requiretoxic precursors and solvents. It is estimated that over 10,000different dyes and pigments are used industrially with over 700,000tonnes of synthetic dye being produced annually worldwide (Chequer etal., 2013, Eco-friendly textile dyeing and finishing, pp. 151-176).

Microbial production of pigments has been studied for hundreds of years.For pigments such as prodigiosin and violacein, naturally occurringmicrobes have been specifically cultured for pigment production(JP10113169; JP55019070A; JP55148091; JP63245666A).

U.S. Pat. No. 4,520,103 to Ensley describes a method for production ofindigo with a recombinant bacterium in a medium that is indole free. Useof specific strains of a recombinant E. coli to produce indigo orindigotin from indole is particularly described using a gene encoding anaromatic dioxygenase from another bacterium to convert the indole.

Indole preparation is described in U.S. Pat. No. 5,112,747 to VanGrinsven et al. Hart et al (Microbiology 138 211-216 (1992) described arecombinant E. coli containing a cloned Rhodococcus gene for producingindigo and indirubin. Indole is produced which is oxidized to indigo.

U.S. Pat. No. 5,077,201 to Eyal et al describes a novel mutant strain ofMorel mushroom which has been found to produce the blue pigment indigoby submerged fermentation in a nutrient culture medium containing acarbon and a nitrogen substrate.

Numerous carbon sources have been evaluated for the microorganismpigmentation including glycerol, maltose, sucrose, citrate, lactose andglucose (World Journal of Microbiology & Biotechnology (2005) 21:969-972). As discussed elsewhere in this document, carbon sources suchas glycerol act as antibacterial agents at concentrations exceeding 10%(v/v). Correspondingly these compounds are used as carbon sources duringmicroorganism pigment production at mild concentration typically <5%(v/v), more commonly 1% (v/v).

Dye Extraction

Once pigments that work as dyes have been produced by the microbe,extraction procedures include the use of organic solvents such aschloroform, ether, ethyl acetate, aqueous sulfuric acid, acetone,hexane, benzene, ethanol or methanol (U.S. Pat. Nos. 5,077,201;5,691,171; Rettori and Duran, 1998, World J. Microbiol. Biotech 14:685-688; JP10113169; JP63245666 A), or boiling the microbe in an aqueoussolution (JP2810287B2; JP10113169). Solvent extraction produces wastechemicals that are difficult and expensive to recycle or dispose of andare highly deleterious to aquatic life if discharged into waterways.

Secondary pigment extraction steps may include volume reduction, solventchange, or sonication or freezing (U.S. Pat. Nos. 5,834,297; 5,691,171).

Fabric Pre-Treatment

U.S. Pat. No. 6,436,696 discloses the treatment of textile fibers withenzymes in the absence of surfactants, with the effect of increasing thewettability and absorbency of the fibers. The enzymes are pectinases,cellulases, proteases, lipases or combinations thereof. The wettingproperties of cotton fibers are found to be most substantially improvedby treatment with a mixture of cellulase and pectinase. As disclosed inU.S. Pat. No. 6,436,696, said enzymes can be produced by microorganismsincluding fungi and bacteria. Microorganisms that produce suitablelipases include Candida ancudensis, Candida Antarctica, Candidaatmaspherica, Candida bombi, Bacillus amyloliquefaciens, Bacillusmegaterium, Bacillus subtilis and many others.

Known fabric pre-treatments have been used in the dyeing process formicrobially produced dyes. Pre-treatments include: 1) standard soap,anhydrous sodium carbonate, L-histidine mono-hydrochloride mono-hydrate,NaCl and NaHPO4 for fabric swelling and removal of impurities and 2)mordants such as alum, copper sulfate, ferrous sulfate, sodium silicate,slaked lime and tamarind preparation (Chequer et al., 2013, Eco-friendlytextile dyeing and finishing, pp. 151-176).

Dye Deposition

Since the 1940s both glycerol and glycerol-derived alkyds have foundwidespread application in many dyeing and printing procedures fortextiles (Uses of Glycerol, compiled by The Glycerol Producers'Association). Glycerol itself produces dyestuff pastes of excellentworkability, promotes the fixation of dyes in printing pastes, increasescolor value in printing and assists in the retention of moisture in theager.

Glycerol is an ingredient of many dyes shipped in paste form since itprevents the dyes from drying out, and sticking to the sides of thedrum. Its non-corrosiveness and low freezing point are desirable in thisapplication. During dyeing, the water miscibility of the glycerolpresent in the dye paste and its solvent action on many types ofdyestuffs aid in dispersing the latter in the dye bath, where the highboiling point of glycerol is another advantage. Occasionally glycerol isadded directly to the dye bath as was the case with the nylon dyeingformulas developed by the Nylon Task Committee during World War II tomeet the washfastness and other requirements of the Quartermaster Corps.In naphthol dyeing, glycerol is sometimes used before coupling toimprove stability of the naphthol solution.

The concentration of glycerol typically used in dye mixtures and dyepastes is limited to minimize the time required to dry fabricspost-dyeing and reduce marking off difficulties respectively(“Technicus” Rayon Textile Mo. 24, 65, August 1943). In one case, to dyeblue shades, the following mixture was used: 1.7 kg ChlorindanthreneBlue; 39.0 litres Sodium Hydroxide; 6.4 kg hyposulfate; and 0.5 kgglycerol (Textile Research Journal March 1944 vol. 14 no. 3 69-73). Herethe glycerol concentration was 1.1% (v/v).

According to Bennett, H., “Chemical Formulary,” Vol. VI, New York, Chem.Publishing Co., pp. 518-9, 1943, a typical textile printing dyecontains: 20 grams direct color; 310 grams hot water; 50 grams glycerol;20 grams sodium phosphate; 500 grams starch-based thickeners; 100 gramsegg albumin. Contemporary acid print pastes prescribed online by RobertGordon University contain 0.1-3 grams acid dyestuff, 5 grams glycerol;20 mls warm water; 60 grams Manutex RS; 2 grams Ammonium Oxalate; and 5mls of hot water. Thus it can be observed that the recommendedconcentration of glycerol contained in fabric printing dyes has remainedat 5% (v/v) for over 70 years.

Other dyeing procedures in which glycerol finds application include thevat dyeing of acetate fabrics, the dyeing of cottons with direct colors,and the preparation of dyeing compounds for use on wool, silk, cotton,synthetic fibers and particularly rayon and staple fibers made fromcellulose. Spray dyeing processes frequently utilize glycerol as asolvent, dispersant and suspending agent for dyes or pigments. Thenonfoaming characteristics of the resulting compositions promote evendyeing. It may also be used to produce blended tints of “umbray”effects. Besides this, it has application in fluid bed dyeing, dispersedacetate dyes and azoic dyes.

Glycerol has also been used as a textile-conditioning agent used widelyin the lubrication, sizing, and softening of yarn or fabric. Itseffectiveness in these and similar applications is due mainly toviscosity and hygroscopicity, both properties contributing to theplasticizing action. Hygroscopic, or humectant, qualities also accountfor the utilization of glycerol in special treatments, such as processesto increase the wearability of fabrics or to prevent static charges onfibers. Because of impermeability to poison gas, particularly “mustard”,glycerol has found application in gas-resistant finishes. Watersolubility is an asset too when glycerol serves as a lubricant. Thisobviates the need for strong scouring agents, which tend to injure thefabric but which must often be used to remove other lubricating oils.

As an additive to lubricating compositions, sizes, or various finishes,glycerol acts as a plasticizer, solvent, and penetrant. It preventsdrying out and caking on the fiber, eliminates the dusting of sizes, andmay aid in dispersing water-insoluble lubricating oils applied from awater bath.

Glycerol is also included in aqueous and solvent-based print inkformulations. Here this substance acts as a humectant to inhibitevaporation of the carrier fluid whilst controlling ink viscosity andthereby fluid dynamics during the deposition process. Ink depositionprocesses may include inkjet printing, screen printing, pad printingetc. Typically the concentration of glycerol plus other humectants inthe ink formulation is <20% (v/v) (Digital Imaging: Water-based Inks andHSE, Photo Marketing Association International, 2004). As explained inU.S. Pat. No. 3,846,141 Jet printing ink composition, higherconcentrations of glycerol or other humectants cannot be used in jetprinting because the ink compound becomes too viscous and consequentlyits passage through the jets becomes inhibited.

WIPO Patent Application WO/2006/019672 describes the inclusion ofglycerol in an eradicable ink formulation. Here glycerol is present inan amount greater than the water content, which is relatively low, andthe eradicable dye is provided in substantial amounts so that theglycerol and eradicable dye provide the requisite viscosity for aball-pen ink. Here water is present in an amount ranging from about 10to about 20% (v/v) and the glycerol is present in an amount ranging fromabout 30 to about 50% (v/v).

In all of the aforementioned circumstances glycerol is used to changethe physical properties, primarily viscosity and hygroscopy, of theaqueous or solvent-based carrier fluid but not on the colorant itselfsuch as azo dye or pigment. More specifically, glycerol has nomeaningful, purposeful or intentional interactions with the colorantdirectly regardless of the presence or absence of a carrier fluid.

U.S. Pat. No. 5,872,002 describes a method of textile patterning usingmicroorganisms to decolorize fabrics previously dyed with an azo dye(which may also contain a non-azo dye). This technique is based on theability of bacterial strains such as Xanthomonas NR25-2 to metabolizeazo dyes comprising a variety of isomers. Using either patterned heatingelements, or patterned acidic pastes, or patterned alkaline pastes, orpatterned disinfectants, regions of bacterial strains deposited onto afabric are deactivated thereby preserving the azo colouring. Notablythese bacterial strains do not produce or deposit pigments themselvesbut remove azo dyes by partially or completely metabolizing a previouslydeposited azo dye.

Extracted microbially produced dyes in solution can be depositeddirectly onto the fabric with a conventional procedure (JP10113169;JP2810287B2). For violacein and prodigiosin, this has been shown to workon a variety of fabric types (Yusof et al., 2012, Application ofbacterial pigments as colorant: the Malaysian perspective).

Glycerol is widely reported as a carbon source for microbe production(Biotechnology Advances 27 (2009) 30-39). Typically the concentration ofglycerol in M9 media is 2% (v/v). Higher concentrations of glycerol tendto inhibit microbial growth. Indeed glycerol has been studied as anantibacterial agent for the long-term preservation of skin allografts(Burns, Volume 34, Issue 2, 205-211).

Dye Fixation

For extracted microbially produced dyes, mordanting (see Fabricpre-treatment) has been shown to improve dye transfer, yielding darkercoloured fabric (Chequer et al., 2013, Ecofriendly textile dyeing andfinishing, pp. 151-176; Yusof et al., 2012, Application of bacterialpigments as colorant: the Malaysian perspective). Some methods includethe addition of post-dyeing mordants such as metal salts. Heat treatmentfor dye fixation is commonly used, with methods including heating insolution at temperatures above 80° C. (JP10113169; Chequer et al., 2013,Eco-friendly textile dyeing and finishing, pp. 151-176).

U.S. Pat. No. 2,297,230 describes the use of glycerol as an additiveduring a textile steam treatment process. Here glycerol serves tostrengthen the binding of Turkey Red oil, a synthetic detergent, intothe fabric during a steam treatment. U.S. Pat. No. 2,297,230 furtherspecifies that glycerol should be dispensed in conjunction with formalin(formaldehyde in water) to prevent to a considerable extent theformation of mildew and spores. Hence U.S. Pat. No. 2,297,230 is notrelevant to this Invention because: (a) it does not relate to textiledyeing, (b) employs steam and not liquid water, and (c) includes asubstance that would be highly deleterious to the microbiologicalprocess specified in this Invention.

Waste Management

All the above steps require waste management and water economy as manydyes, their precursors and the solvents used to produce and extract themare hazardous to human health and the environment. The textile industryconsumes a substantial amount of water in its manufacturing processes,mainly due to the dyeing process. Waste water from textile plants isclassified as the most polluting of all industrial processes (Chequer etal., 2013, Eco-friendly textile dyeing and finishing, pp. 151-176).

In the dyeing process, 10-50% of the dye is lost as waste and ends up inthe effluent (Chequer et al., 2013, Eco-friendly textile dyeing andfinishing, pp. 151-176). On a global scale, this results in 2×10⁵ tonnesof dye being released into the environment annually (Chequer et al.,2013, Eco-friendly textile dyeing and finishing, pp. 151-176). Onaverage, the ratio of the required water-to-fabric mass is up to 100:1(Huntsman Textile Effects, Singapore).

In conclusion, existing methods for producing fabric dyes andtransferring and fixing said dyes to fabrics, yarns and textilesnecessitates the production and usage of toxic chemicals and thegeneration of toxic wastes. Furthermore the consumption of water duringdyeing processes and subsequent wastewater treatment impose significantburdens on local water demand. Whilst the merits of glycerol as a dyeadditive and fabric treatment agent are widely acknowledged,concentrations of this substance are limited to low levels (typicallyless than 5% (v/v). Whilst microbiological production of fabric dyes hasbeen discussed, such methods do not extend to improved methods forextracting, depositing and fixing said dyes into the fabric yarns.

SUMMARY OF INVENTION

This process can be achieved by observing the conditions below whendyeing substrates (such as fabrics) directly using microorganisms thatact as agents for substrate pretreatment as well dye production,deposition and fixation.

For dye production, both natural (non-recombinant) microorganismscapable of producing both intermediate and end point pigments as well asrecombinant microorganisms which have been modified so as to be able toproduce either intermediate or end point pigments can be used in thisprocess.

Pre-treatment (including substrate modification of any type) occurs viathe action of metabolic processes conducted by microorganisms permeatingthe substrate and by the medium compositions which facilitate theseprocesses. Pre-treatment allows for more effective dyepenetration/permeation/fixation in the substrate. Dye deposition isachieved via localized production and release of the dye by themicroorganism which have permeated the substrate. Increased localconcentrations lead to higher dye uptake and the substantial reductionof large amounts of free dye in solution lead to a substantially reducedamount of waste product. In the dye fixation step, the vast majority ofdye present in the inactivated microorganism is transferred to thesubstrate due to lysis.

Dye fixation is achieved via exposure of the treated substrate totemperatures exceeding 121° C. This has the dual purpose of inactivatingall microorganisms present on the substrate as well as fixation of thedye to the substrate. Industrial automatic autoclaves machine, made bySparrow Tex Engineering Works and Bluemoon Machines ManufacturingCompany, can be used for heat setting and conditioning of yarn invarious capacity ranges.

A final wash step removes the vast majority of inactivatedmicroorganisms and microorganism related detritus from the substrate.The final substrate has then been sterilized and cleaned to a standardnearing medical device requirements.

Advantageously the described methods surpass conventional methods of dyeproduction, dye transfer and dye fixation in regards to wastegeneration, water consumption, and energy consumption. Wastes generatedusing the described process do not include any of the following: organicsolvents, concentrated acidic or alkali products, such as bleach.Handling, inactivation and disposal of the waste products deriving fromthis process becomes safe and inexpensive in comparison to conventionalmethods. All waste products generated from this process arebiodegradable compounds. Some of the waste products generated from thisprocess may have commercial value, for example as a plant fertilizer.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood with reference to the followingdrawings and description.

FIG. 1A illustrates the first step of the method for growing anddepositing pigments produced by microorganisms according to JP2810287B2showing a pigment-producing bacteria species inoculated in a mediasolution.

FIG. 1B illustrates the second step of the method for growing anddepositing pigments produced by microorganisms according to JP2810287B2showing bacteria inoculated in a second media solution.

FIG. 1C illustrates the third step of the method for growing anddepositing pigments produced by microorganisms according to JP2810287B2showing a wool decomposition product added to the media solution.

FIG. 1D illustrates the fourth step of the method for growing anddepositing pigments produced by microorganisms according to JP2810287B2showing threads added to the media solution and some of the pigmentscontained within the microorganisms released into the media solution.

FIG. 1E illustrates the fifth step of the method for growing anddepositing pigments produced by microorganisms according to JP2810287B2showing threads removed from the dye bath and washed under runningwater.

FIG. 2A illustrates a method for dyeing textiles according to thepresent invention showing a dye-producing microorganism inoculated in avolume of media solution.

FIG. 2B illustrates a method for dyeing textiles according to thepresent invention showing a culture medium containing dye-containingmicroorganisms, a volume of media solution, a second volume of carbonsource and a substrate.

FIG. 2C illustrates a method for dyeing textiles according to thepresent invention showing dye-containing microorganisms adsorbed into asubstrate.

FIG. 2D illustrates a method for dyeing textiles according to thepresent invention showing a dyed substrate being removed from a culturemedium.

FIG. 2E illustrates a method for dyeing textiles according to thepresent invention showing a dyed substrate being subjected to a heattreatment.

FIG. 2F illustrates a method for dyeing textiles according to thepresent invention showing a washing machine removing lysedmicroorganisms from a substrate having dye molecules fixed to itsfibers.

FIG. 3 is a micrograph showing E. coli grown in a liquid culture in a 1%(v/v) concentration of glycerol in 50% (v/v) LB medium and water.

FIG. 4 is a micrograph showing E. coli grown in a liquid culture in a 5%(v/v) concentration of glycerol in 50% (v/v) LB medium and water.

FIG. 5 is a micrograph showing E. coli grown in a liquid culture in a 5%(v/v) concentration of glycerol in 50% (v/v) LB medium and water.

FIG. 6 is a micrograph showing E. coli grown in a liquid culture in a10% (v/v) concentration of glycerol in 50% (v/v) LB medium and water.

FIG. 7 is a micrograph showing E. coli grown in a liquid culture in a20% (v/v) concentration of glycerol in 50% (v/v) LB medium and water.

FIG. 8 is a micrograph showing E. coli grown in a liquid culture in a20% (v/v) concentration of glycerol in 50% (v/v) LB medium and water.

FIG. 9 is a micrograph showing E. coli grown in a liquid culture in a50% (v/v) concentration of glycerol in 50% (v/v) LB medium and water.

FIG. 10 is a micrograph showing E. coli grown in a liquid culture in a50% (v/v) concentration of glycerol in 50% (v/v) LB medium and water.

FIG. 11 is a micrograph showing E. coli grown in a liquid culture in a50% (v/v) concentration of glycerol in 50% (v/v) LB medium and water.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The features and advantages of the present invention will be morereadily understood, by those of ordinary skill in the art, from readingthe following detailed description. It is to be appreciated thosecertain features of the invention, which are, for clarity, describedabove and below in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention that are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany sub-combination. In addition, references in the singular may alsoinclude the plural (for example, “a” and “an” may refer to one, or oneor more) unless the context specifically states otherwise.

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about.” In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, the disclosure of these ranges is intended as a continuous rangeincluding every value between the minimum and maximum values.

All patents, patent applications and publications referred to herein areincorporated by reference in their entirety.

This process aims to combine substrate pre-treatment and localizedproduction, deposition and fixation of substrates (such as fabrics).Fabrics can be natural (cotton, silk, wool and others of a similarnature) or synthetic (polyester, rayon, elastaine and others of asimilar nature) in composition.

Dye production is achieved via use of a microorganism capable ofproducing pigments with properties desired in a dye. Both natural(non-recombinant) microorganisms capable of producing both intermediateand end point pigments and recombinant microorganisms which have beenmodified so as to be able to produce either intermediate or end pointpigments can be used in this process. Examples include but are notlimited to: Serratia spp, Janthinobacter spp., Chromobacterium spp.,Bacillus spp., Escherichia spp., Cyanobacterium spp., Pseudomonas spp.As an example, a K12 derivative of E. coli has been modified to producehigh quantities of violacein by introducing a plasmid that carries are-factored violacein operon. The re-factoring includes a re-arrangementof the order of enzyme coding sequences, the addition of ribosomebinding sites in front of each coding sequence and codon optimisation ofthe coding sequences for expression in E. coli.

Virtually any microorganism which can be genetically modified may beincorporated into this process. The innovative step regarding thisprocess is in the direct application of the microorganism to thesubstrate in order to facilitate localized dyeing of the substrate.

Pre-treatment (including substrate modification of any type) occurs viathe action of metabolic processes conducted by microorganisms permeatingthe substrate and by the medium compositions which facilitate theseprocesses. Pre-treatment allows for more effective dye penetration,permeation and fixation in the substrate.

The pre-treatment step is performed by the microorganism in a wide rangeof conditions which drive microorganism metabolism via variousparameters, such as nutrient content, pH and salinity. The nature of themicroorganism employed in this process will dictate the optimumcomposition of the medium. As an example, Chromobacterium violaceumrequires a very different optimal environment than Escherichia coli.

As an example of embodiment of this invention, a K12 derivative of E.coli which has been modified to produce high quantities of violacein wasused to pre-treat the following substrates: silk, wool, rayon,polyester, elastaine, cotton and flax. The medium compositions used topre-treat the substrate with the above mentioned strain of E. coliinclude the following base components: carbon source, nitrogen source,amino acid source, metal salt source and water.

Explanation

Without wishing to be constrained by theory, we believe the improvedadsorption of dye-containing microorganisms into the textile fabric iscaused by changes in the morphology of these microorganisms when exposedto a carbon source when this carbon source is beyond a thresholdconcentration. More specifically, beyond a certain concentration ofcarbon source the microorganisms become significantly longer anddistorted in shape. Longer microorganisms are more likely to becomeentangled in the fabric yarns whilst contortions along their lengthmeans they are harder to be dislodged from their anchoring amongst thefibers. The precise concentration threshold will depend on numerousfactors such as the microorganism species, the carbon source used, andoperating conditions such as temperature and pH level. Generallyspeaking we have found the threshold concentration is in the range 10%(v/v) to 60% (v/v), more commonly 20% (v/v) to 40% (v/v).

Figures

FIG. 1 shows a method for growing and depositing pigments produced bymicroorganisms according to JP2810287B2. FIG. 1A shows a first stepwhere a single pigment-producing bacteria species (1) is inoculated in amedia solution (2). FIG. 1B shows a second step where the bacteria areinoculated in a second media solution (3) for 18 hours at 30° C. FIG. 1Cshows a third step where a wool decomposition product (4) is added tothe media solution and shaken for five days at 30° C. FIG. 1D shows afourth step where threads (5) are added to the media solution whilstsome of the pigments contained within the microorganisms are releasedinto the media solution (6) by boiling the media solution at 100° C. for20 minutes. FIG. 1E shows a sixth step where the threads are removedfrom the dye bath after boiling and washed under running water to removeresidual media solution and loose pigments. An unspecified quantity ofpigments shall remain in the media solution (7) and attached to the wooldecomposition product (8).

FIG. 2 shows the method for dyeing textiles according to this invention.Before the first dyeing step, dye-producing microorganisms (10) areprepared according to standard microbiology methods. These methods mayinclude techniques such as synthetic biology and genetic engineering. Asingle colony of the microorganism is inoculated in media solutionaccording to standard methods.

In a first dyeing step, the dye-producing microorganism (10) isinoculated in a volume of media solution (11) and allowed to growovernight according to standard microbiology techniques. The resultingculture medium containing high concentrations of dye-containingmicroorganisms (12) is then supplemented with a volume of media solution(13) and a second volume of carbon source (14). The resultingconcentration of carbon source should be in the range 10% (v/v) to 90%(v/v), preferably 15% (v/v) to 60% (v/v), more preferably 20% (v/v) to40% (v/v). A substrate (15), i.e. fabric or yarn, is added to theculture and incubated overnight according to standard methods.

After overnight incubation the vast majority of dye-containingmicroorganisms will be adsorbed into the substrate (16) including thespaces between neighboring fibers. Correspondingly the culture media andcarbon source solution (17) shall be substantially bereft ofmicroorganisms.

In a second dyeing step, the dyed substrate (18) is removed from theculture medium and washed in a water bath (19) to remove residual carbonsources and microorganism detritus from the substrate. Waste wash water,which may contain residual quantities of free microorganisms, can bereused in subsequent dyeing batches or sterilized in a mild bleach orsteam autoclave. The dyed substrate is subjected to a heat treatmentstep such dry ironing (not shown) or steam autoclaving (20) or microwave(not shown). In all cases the applied temperature should be higher than100° C., preferably 121° C. This second dyeing step performs the dualrole of (a) releasing the dye from microorganisms directly onto thesubstrates onto which the microorganisms have become adsorbed throughlysation, and (b) fixing dye release with the lysate onto the substrate.Dye fixation and sterilization of waste wash water can be performed inthe same autoclave cycle.

A washing machine (21) operating using standard settings (for example40° C. wash using bio detergent) removes lysed microorganisms (22) fromthe substrate (23) with dye molecules (24) fixed to its fibers. Thewashed substrate is dried using standard methods (not shown).

FIG. 3 shows the growth of a microorganism in liquid culture, here E.coli, in a 1% (v/v) concentration of glycerol in 50% (v/v) LB medium andwater. E. coli was grown for 24 hours in 50% (v/v) LB medium (10 g NaCl,5 g Yeast Extract, 1 g Peptone, 1 L water) and 1% (v/v) glycerol. Themicroorganism has grown successfully and the measured average bacterialength is approximately 3.5 microns.

FIG. 4 and FIG. 5 show the growth of a microorganism in liquid culture,here E. coli, in a 5% (v/v) concentration of glycerol in 50% (v/v) LBmedium and water. E. coli was grown for 24 hours in 50% (v/v) LB medium(10 g NaCl, 5 g Yeast Extract, 1 g Peptone, 1 L water) and 5% (v/v)glycerol. The microorganisms have grown successfully though not as muchas the 1% (v/v) concentration. The measured average bacteria length isin the range 4 microns to 5 microns.

FIG. 6 shows the growth of a microorganism in liquid culture, here E.coli, in a 10% (v/v) concentration of glycerol in 50% (v/v) LB mediumand water. E. coli was grown for 24 hours in 50% (v/v) LB medium (10 gNaCl, 5 g Yeast Extract, 1 g Peptone, 1 L water) and 10% (v/v) glycerol.Microorganism growth is substantially lower than the 1% (v/v) case. Themeasured average microorganism length is 11 microns. Microorganisms havebecome curved along their length.

FIG. 7 and FIG. 8 show the growth of a microorganism in liquid culture,here E. coli, in a 20% (v/v) concentration of glycerol in 50% (v/v) LBmedium and water. E. coli was grown for 24 hours in 50% (v/v) LB medium(10 g NaCl, 5 g Yeast Extract, 1 g Peptone, 1 L water) and 20% (v/v)glycerol. Microorganism growth is very substantially lower than the 1%(v/v) case. The measured bacteria length is in the range 15 microns to20 microns. Microorganisms have become substantially curved along theirlength.

FIG. 9 to FIG. 11 show the growth of a microorganism in liquid culture,here E. coli, in a 50% (v/v) concentration of glycerol in 50% (v/v) LBmedium and water. E. coli was grown for 24 hours in 50% (v/v) LB medium(10 g NaCl, 5 g Yeast Extract, 1 g Peptone, 1 L water) and 50% (v/v)glycerol. Microorganism growth is very substantially lower than the 1%(v/v) case. The measured bacteria length is in the range 10 microns to15 microns. Microorganisms have become substantially warped along theirlength.

Employable Media Additives:

Medium compositions involved the addition of salts examples of whichinclude, but are not limited to: NaCl, KCl, CaCl2, MgCl2, MnCl2, ZnCl2,alone or in combination. Medium compositions involved the addition of anamino acid source, examples of which include, but are not limited to:Tryptone, Peptone, Bacto-peptone, Casein-amino acids, alone or incombination. Medium compositions involved the addition of a carbonsource, examples of which include, but are not limited to: Yeastextract, Sucrose, Glucose, Glycerol, Fructose, Xylose, Lactose,Arabinose, alone or in combination. Medium compositions involved theaddition of a nitrogen source, examples of which include, but are notlimited to: yeast extract, Tryptone, Peptone, Bacto-peptone,Casein-amino acids, alone or in combination. All of the above mediumadditives may be employed with varying results, depending on theorganism used for the pre-treatment process.

Growth Conditions:

Optimal growth conditions vary with the microorganism employed for thepre-treatment process. Parameters which greatly affect the end resultinclude but are not limited to: pH, Salinity and Temperature.

As an example of the embodiment of this claim, a K12 derivative of E.coli which has been modified to produce high quantities of violacein wasused to pre-treat the following substrates: silk, wool, rayon,polyester, elastaine and cotton. Varied growth conditions were tested,with pH ranges between 5-9, salinity ranges of 0.1% to 3%, andtemperatures between 20° C. and 42° C. Optimum ranges for theseparameters were found to be: pH 5.8-8.2, salinity 0.5%-1.5% andtemperature: 30° C. to 40° C.

Pre-treatment facilitates the interaction between the dye-containingvessel (microorganism) and the substrate (fabric). To achieve thiseffect, the employed microorganism is grown in a suitable medium (seemedium composition above) for a period of 12-48 hours depending on theinoculant-to-inoculate ratio, medium composition employed and growthconditions used. The entire culture is then supplemented with additionalmedium (see medium composition above) and the substrate is added at thispoint. Pre-treatment takes place within a similar time frame as thegrowth period.

Dye deposition is achieved via localized production and release of thedye by the microorganisms which have permeated the substrate. Increasedlocal concentrations lead to higher dye uptake and the absence of largeamounts of free dye in solution lead to a substantially reduced amountof waste product. Dye deposition rates will vary depending on whichpigment is being produced as well as what microorganism is beingemployed.

These parameters will vary with the cytotoxicity of the pigment producedby the microorganism, the water solubility of the pigment, the pigmentaffinity for the substrate and the growth conditions employed (seegrowth conditions above). As an example of embodiment of this claim, aK12 derivative of E. coli which has been modified to produce highquantities of violacein was used to deposit dyes on/in the followingsubstrates: silk, wool, rayon, polyester, elastaine, cotton and flax.Nearly complete penetration and association of theviolacein-producing/containing E. coli with the substrate was observedwithin two hours post substrate addition to the supplemented medium andcontinues throughout the substrate incubation period.

Finishing Step:

A final finishing step is achieved via exposure of the treated substrateto temperatures exceeding 121° C. This has the dual purpose ofinactivating all microorganisms present on the substrate as well asfixation of the dye to the substrate.

The vast majority of dye present in the inactivated microorganism istransferred to the substrate due to lysis. The finishing step theninvolves a final wash, which removes the vast majority of inactivatedmicroorganisms and microorganism related compounds from the substrate.The final substrate has then been sterilized and cleaned to a standardnearing medical device requirements.

Waste Products:

Unincorporated dye (dye waste) is below the industry standard of 3% anda level of incorporation has been achieved by the invented process thatsurpasses 99.997% efficiency. Spent medium such as the dissolved salts,amino acids and carbon sources, remaining after the dyeing process canbe recycled and re-used for subsequent dyeing processes using either thesame or a different microbiological dye. Thus via at least tworoutes—firstly less water use during the initial dyeing process andsecondly the ability to re-use spent media solutions withouttreatment—water use is reduced in the described methods when compared toconventional methods.

1. A method for dyeing a substrate, comprising: a. culturing adye-producing microorganism in the presence of a substrate to be dyed,and in the presence of a growth medium comprising a carbon source abovea predetermined threshold concentration, such that the microorganism iscultured in contact with the substrate; b. lysing the culturedmicroorganism to release dye in contact with the substrate; and c.fixing the released dye onto the substrate.
 2. The method of claim 1,wherein the predetermined threshold concentration of carbon source is10% (v/v) to 90% (v/v) depending on the carbon source selected tooptimize the transfer rate of dye-containing microorganisms to thesubstrate, and the quality of subsequent dye fixation to the substrate.3. The method of claim 1, wherein the lysing and fixing are carried outin a single process.
 4. The method of claim 1, wherein the lysing andfixing are carried out by exposing the substrate and microorganism toheat above 101° C.
 5. The method of claim 1, wherein one or more cultureparameters selected from temperature, carbon dioxide concentration, pHand agitation frequency are selected so as to optimize the production ofdye, the transfer rate to the substrate, and the quality of fixation tothe substrate.
 6. The method of claim 1, further comprising initiallyculturing the dye-producing microorganism in the absence of thesubstrate to be dyed, prior to step 1a.
 7. The method of claim 1,further comprising washing the dyed substrate to remove wastecontaminants prior to and post step 1c.
 8. The method of claim 1,wherein two or more different dye-producing microorganism species areused simultaneously.
 9. The method of claim 1, wherein two or moredifferent dyes are produced by one or more different microorganisms. 10.The method of claim 1, wherein additional dyes, including dyes addedexogenously, are present during the lysing and fixing.
 11. The method ofclaim 1, wherein the substrate is selected from natural, synthetic,semi-synthetic and mixed substrates.
 12. The method of claim 11, whereinthe substrate comprises a member selected from silk, cotton, flax, wool,and leather.
 13. The method of claim 11, wherein the substrate comprisesa member selected from rayon and acetate.
 14. The method of claim 11,wherein the substrate comprises a member selected from polyester, nylon,acrylic, elastin, polyvinyl and similar petrochemical derivatives. 15.The method of claim 1, wherein the dye-producing microorganism producesa dye selected from biologically derived pigments, chromoproteins,fluorescent proteins and bioluminescent proteins.
 16. The method ofclaim 1, wherein the microorganism is a eukaryotic organism.
 17. Themethod of claim 16, wherein the eukaryotic organism is selected fromplant, algae, fungi, worms and arthropods.
 18. The method of claim 1,wherein the microorganism is a prokaryotic organism.
 19. The method ofclaim 18, wherein the prokaryotic organism is selected from archae andeubacteria.
 20. The method of claim 18, wherein the prokaryoticorganisms are Gram positive bacteria selected from the group consistingof Bacillus spp. and Clostridium spp. 21-36. (canceled)